Previous lectin-histochemical and immunocytochemical investigations using fixed tissue revealed perineuronal nets as lattice-like accumulations of extracellular matrix proteoglycans at the surface of several types of neurons. In the present study, perineuronal nets in the rat brain were labelled for the first time in vivo by stereotaxic injections of biotinylated Wisteria floribunda agglutinin (Bio-WFA), as well as in vitro, by incubation of unfixed brain slices with the same lectin. Six days after Bio-WFA injections into the parietal cortex, medial septum, reticular thalamic nucleus and red nucleus, the lectin remaining bound to perineuronal nets was detected by streptavidin/biotinylated peroxidase complexes or red fluorescent Cy3-streptavidin, respectively. Double-fluorescence labelling showed that Bio-WFA applied in vivo reacted with the chondroitin sulphate proteoglycan immunoreactive perineuronal nets in the injection zone. Labelling of perineuronal nets in unfixed slices was obtained with either Cy3-tagged WFA or Bio-WFA and subsequent visualization by Cy3-streptavidin which confirmed the region-dependent distribution patterns and the structural characteristics of perineuronal nets known from histochemical studies. These results provide support for the role of extracellular matrix proteoglycans to maintain a considerable chemical and, probably, spatial heterogeneity of the extracellular space in vivo. The ability of in vivo and in vitro labelling may promote the functional characterization of the extracellular matrix in various brain structures including its species-dependent neuronal association patterns.